Preparations of biological and chemical origin including, but not limited to, preparations from bacteria, viruses, yeast, and plants have been used to stimulate or to inhibit responsive cells in an animal, including a human. Among bacteria, preparations of cell wall from, but not limited to, Mycobacterium species have been used to treat diseases including, but not limited to, skin diseases U.S. Pat. No. 4,340,586), bacterial infections (U.S. Pat. No. 3,172,815), viral infections (U.S. Pat. No. 4,744,984) and cancers (U.S. Pat. No. 4,503,048). Mycobacterial cell wall extracts are composed primarily of peptidoglycan and glycolipid (Chin et al. Journal of Urology 156:1189-1193, 1996) and contain N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) and mycolic acid derivatives. Both muramyl dipeptide and mycolic acid derivatives stimulate the immune system by activation of macrophage and monocyte mediated reactions (Mallick et al. Comparative Immunology and Microbiology of Infectious Diseases 8:55-63, 1985; Teware et al. Veterinary Parasitology 62:223-230, 1996). As used herein, the immune system is defined to include macrophages, monocytes, lymphocytes and leukocytes. These reactions, mediated by the immune system, induce cytolysis, which is the complete or partial destruction of a cell. However, the therapeutic benefits obtained using such cell wall extracts to treat cancer cells are variable and inconsistent, and appear to depend on the method by which the preparation is prepared and delivered, and on the stability of the preparation.
Activated macrophages and monocytes produce bioactive molecules that initiate, accelerate, amplify and modulate responsive cells of the immune system. By produce is meant synthesize and release. These bioactive molecules include, but are not limited to, cytokines and reactive oxygen species. Cytokines include, but are not limited to, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12) and GM-CSF. IL-12 alone, and in combination with other cytokines, promotes the maturation of leukocytes including, but not limited to, B-lymphocytes, CD4+ T-cells, CD8+ T-cells, and NK cells and induces the secretion of interferon-gamma. The cytokine IL-12 is reported to have anti-cancer activity is some cancer cells lines (Stine et al. Annals NY Academy of Science 795:420-421, 1996; Chen et al. Journal of Immunology 159:351-359, 1997). This anti-cancer activity includes, but is not limited to, activation of specific cytolytic T-lymphocytes, activation of natural killer (NK) cells and induction of the anti-angiogenic proteins IP-10 and MiG. IP-10 inhibits cancer growth and metastasis, inhibits cancer-induced neovascularization, and further activates NK cells (Angillo et al. Annals NY Academy of Sciences 795:158-165, 1996). The cytokine GM-CSF is reported to have pro-cancer activity is some cancer cells lines (Hawkyard et al. Journal of Urology 150:514-518, 1993).
Reactive oxygen species include, but are not limited to, nitric oxide, superoxide radicals, and hydroxyl radicals. Nitric oxide, superoxide radicals, and hydroxyl radicals, among other activities, induce cytolysis and apoptosis in susceptible target cells.
Apoptosis is a genetically programmed, non-inflammatory, energy-dependent form of cell death in tissues including, but not limited to, adult tissues (Steller, H. Science 267:1445-1449, 1995). Apoptosis can be initiated by ligands which bind to cell surface receptors including, but not limited to, Fas (CD95) (French et al. Journal of Cell Biology 133:335-343, 1996) and tumor necrosis factor receptor 1 (TNFR1). FasL binding to Fas and TNF binding to TNFR1 initiate intracellular signaling resulting in the activation of cysteine aspartyl proteases (caspases) that initiate the lethal proteolytic cascade of apoptosis execution (Muzio et al. Cell 85:817-827, 1996), which is associated with nuclear DNA-fragmentation, release of nuclear matrix proteins (NuMA) and loss of cell substrate contact.
Apoptosis also can be induced by intracellular proteins including, but not limited to, p53/p21. p53/p21 act as a transcription factors to induce expression of apoptosis-mediating genes, including, but not limited to, genes encoding proteins that generate free radicals that, in turn, damage the cell's mitochondria, whose contents leak out into the cytoplasm and activate apoptotic caspases (Polyak et al. Nature 389:300-305, 1997).
Cancer is an aberrant net accumulation of atypical cells, which results from an excess of proliferation, an insufficiency of apoptosis, or a combination of the two. Mutations in apoptosis-related genes such as, but not limited to, Fas, TNFR1 and p53/p21 each have been implicated in the pathogenesis of cancers (Levine, A. Cell 88:323-331, 1997; Fisher, D. Cell 78:529-542, 1994). Apoptosis is important not only to the pathogenesis of cancers, but also to their likelihood of resistance to anti-cancer therapies.
Resistance to apoptosis induction has emerged as an important category of multiple drug resistance (MDR), one that likely explains a significant proportion of treatment failures. MDR, the simultaneous resistance to structurally and functionally unrelated chemotherapeutic agents, can be both inherent and acquired. That is, some cancers never respond to therapy, whereas other cancers, initially sensitive to therapy, develop drug resistance. As chemotherapeutic agents rely primarily on induction of apoptosis in cancer cells for their therapeutic effect, drug resistance, which diminishes the effectiveness of chemotherapeutic agents, leads directly or indirectly to reduced apoptosis and is generally associated with poor prognosis in a variety of cancers.
Prior art anti-cancer agents have proven to be less than adequate for clinical applications. Many of these agents are inefficient (Bischoff et al. Science 274:373-376, 1996) or toxic, have significant side effects (Lamm et al. Journal of Urology 153:1444-1450, 1995), result in development of drug resistance or immunosensitization, and are debilitating for the recipient. Moreover, many of these agents depend on Fas, TNFR1 or p53/p21 for their effectiveness.
Therefore, there is a need for a novel therapeutic agent that stimulates responsive cells of the immune system to produce cytokines and reactive oxygen species and which inhibits proliferation of cancer cells and induces apoptosis in cancer cells. This therapeutic agent should be useful as an anti-cancer agent and as an adjunct to other anti-cancer agents. By adjunct is meant useful with other anti-cancer agents to increase treatment effectiveness. Moreover, such a therapeutic agent should be simple and relatively inexpensive to prepare, its activity should be reproducible among preparations, its activity should remain stable over time, and its effects on cancer cells should be achievable with dose regimens that are associated with minimal toxicity.